Neuronal division, survival and differentiation are dependent during development on a diverse group of protein and peptide growth factors. Included in this group of regulatory molecules are recognized trophic factors, such as nerve growth factor (NGF) (Levi-Montalcini, Differentiation, 13:51-53 (1979)), ciliary neurotrophic factor (CNTF) (Lin et al., Science 24:1023-1025 (1989)), fibroblast growth factor (FGF) (Wallicke et al., J. Neurosci. 11:2249-2258 (1991)), insulin-like growth factors 1 and 2 (IGFs 1 and 2) (Ishii et al., Pharmacol. & Ther. 62:125-144 (1994)), brain derived neurotrophic factor (BDNF) (Laibrock et al., Nature 341:149-152 (1989)), glial derived neurotrophic factor (GDNF) (Lin et al., Science 260:1130-1132 (1993)), and neurotrophin-3 and neurotrophin-4/5 (Henderson et al., Nature 363:266-269 (1993)). In addition, cytokines also have neurotrophic properties (Brenneman et al., J. Neurochem. 58:454-460 (1992); Patterson, Curr. Opin. Neurobiol. 2:91-97 (1992)). Although many of the classic growth factors were first recognized as playing important trophic roles in neuron/target cell interactions, it is now clear that glial cells in the central nervous system (CNS) express most of these growth factors/cytokines, and that these support cells play significant roles during development and nerve repair/regeneration.
In this regard, efforts have been made to understand the role of neuropeptides in regulating the release/expression of glia-derived trophic substances and to identify new glial molecules that contribute to the survival of developing CNS neurons. In particular, efforts have been made to understand the role of trophic support for activity-dependent neurons in the CNS. The activity-dependent neurons are a class of neurons that die during electrical blockade due to a reduction of soluble trophic materials in their environment (Brenneman et al., Dev. Brain Res. 9:13-27 (1993); Brenneman et al., Dev. Brain Res. 15:211-217 (1984)). Electrical blockade has been demonstrated to inhibit the synthesis and release of trophic materials into the extracellular milieu of CNS cultures (Agostan et al., Mol. Brain. Res. 10:235-240 (1991); Brenneman et al., Peptides 6(2):35-39 (1985)). Included in this trophic mixture is vasoactive intestinal peptide (VIP) (Brenneman et al., Peptides, supra (1985); Brenneman et al., Proc. Natl. Acad. Sci. USA 83:1159-1162 (1986)).
The 28-amino acid peptide VIP (Said et al., Ann. NY Acad. Sci. 527:1-691 (1988)), has been associated with cellular protection in sensory neurons, axotomized sympathetic neurons and acutely injured lung and airways (see, e.g., Gressens et al., J. Clin. Invest. 100:390-397 (1997)). Indeed, the lack of regulation of VIP expression observed in these injured or inflamed systems probably represents an adaptive response that limits damage and promotes recovery.
VIP has been shown to interact with high affinity receptors present on glial cells (Gozes et al., J. Pharmacol. Exp. Therap. 257:959-966 (1991)), resulting in the release of survival-promoting substances (Brenneman et al., J. Cell. Biol. 104:1603-1610 (1987); Brenneman et al., J. Neurosci. Res. 25:386-394 (1990)), among which are a glial-derived cytokine IL-1-α ((Brenneman et al., J. Neurochem. 58:454-460 (1992); Brenneman et al., Int. J. Dev. Neurosci. 13:137-200 (1995)), and protease nexin I, a serine protease inhibitor (Festoff et al., J. Neurobiol. 30:255-26 (1995)). However, the neuronal survival-promoting effects of the VIP-conditioned medium were observed at very low concentrations that could not be attributed to IL-1 or protease nexin I released from astroglia. Therefore, efforts have been made to identify other survival-promoting proteins released from glial cells stimulated by VIP.
In doing so, a novel neuroprotective protein secreted by astroglial in the presence of VIP was discovered (Brenneman & Gozes, J. Clin. Invest. 97:2299-2307 (1996); Gozes & Brenneman, J. Molec. Neurosci. 7:235-244 (1996)). The neurotrophic protein was isolated by sequential chromatographic methods combining ion exchange, size separation and hydrophobic interaction. This neuroprotective protein (mol. mass, 14 kD and pI, 8.3±0.25) was named Activity Dependent Neurotrophic Factor (ADNF or ADNF I) for two reasons: (1) a blockade of spontaneous electrical activity was necessary to detect the neuroprotective action of this substance in dissociated spinal cord cultures; and (2) VIP, a secretagogue for ADNF, was released during electrical activity, making the presence of ADNF in the extracellular milieu indirectly dependent on spontaneous activity. ADNF was found to exhibit neuroprotection at unprecedented concentrations. More particularly, femtomolar concentrations of ADNF were found to protect neurons from death associated with a broad range of toxins, including those related to Alzheimer's disease, the human immunodeficiency virus (HIV), excitotoxicity, and electrical blockade (see, e.g., Gozes et al., Dev. Brain Res. 99:167-175 (1997)).
During the course of studies directed to the structural characteristics of ADNF, an active peptide fragment of ADNF was discovered. This active peptide, 9-amino acids derived from ADNF (ADNF-9), was found to have strong homology, but not identity, to an intracellular stress protein: heat shock protein 60 (hsp60). Another peptide, ADNF-14, which comprises ADNF-9, was also found to be active, as were other derivatives of ADNF-9. Moreover, ADNF-9 was shown to mimic the potency of the parent protein, while exhibiting a broader range of effective concentrations as compared to the parent protein. In addition, ADNF-9, like ADNF, has been shown to prevent neuronal cell death associated with the envelope protein (gp120) from HIV (see Dibbern et al., J. Clin. Invest. 99:2837-2841 (1997)), with excitotoxicity (N-methyl-D-aspartate), with the β-amyloid peptide (putative cytotoxin in Alzheimer's disease), and with tetrodotoxin (electrical blockade) (see Brenneman & Gozes, J. Clin. Invest. 97:2299-2307 (1996)).
The discovery of ADNF has provided additional knowledge regarding the neuroprotective action of VIP (Gozes & Brenneman, Mol. Neurobiol. 3:201-236 (1989); Said, J. Clin Invest. 97:2163-2164 (1996)). Moreover, the neurotrophic properties of the ADNF polypeptide have significant therapeutic and diagnostic implications. The discovery that ADNF activity can be mimicked by a 9-amino acid peptide is predicted to facilitate innovative drug design for the treatment of the neurological symptoms associated with HIV infection, Alzheimer's disease, and other prevalent neurodegenerative diseases. Although ADNF, ADNF-9, and ADNF-14 have unlimited potential as neuroprotectants, it would still be advantageous to identify other survivalpromoting proteins released from glial cells stimulated by VIP.